Actinomyces ruminis sp. nov., an obligately anaerobic bacterium isolated from the rumen of cattle

An obligately anaerobic, rod-shaped, Gram-stain-positive, non-spore-forming, non-motile bacterial strain; designated as CtC72T was isolated from the rumen of cattle. The 16S rRNA gene sequence similarity of less than 98.65% revealed the strain as a member of the genus Actinomyces, nearest to but distinct from Actinomyces qiguomingii DSM 106201T, Actinomyces ruminicola DSM 27982T, Actinomyces procaprae JCM 33484T, Actinomyces succiniciruminis TISTR 2317, Actinomyces glycerinitolerans TISTR 2318. The low values of digital DNA-DNA hybridization (< 70%) and average nucleotide identity (< 95%) further highlighted the distinctive nature of strain CtC72T from its closest relatives. The strain CtC72T could grow at temperatures between 30 and 50 °C (optimum 40 °C), pH between 6.0 and 9.0 (optimum 7.5–8.0), and NaCl between 0 and 1.5% (optimum 0%). The strain hydrolysed cellulose and xylan and utilised a range of mono-, di-, and oligo-saccharides as a source of carbon and energy. Glucose fermentation resulted in acetic acid and formic acid as major metabolic products, while propionic acid, lactic acid, and ethanol as minor products along with CO2 production. The DNA G + C content of strain CtC72T was 68.40 (mol%, Tm) and 68.05 (%, digital). Major cellular fatty acids (> 10%) were C16:0, C18:1 ω9c, and C18:1 ω9c DMA. Based on these data, we propose that strain CtC72T be classified as a novel species, Actinomyces ruminis sp. nov., under the genus Actinomyces. The type strain is CtC72T (= KCTC 15726T = JCM 32641T = MCC 3500T).


Introduction
The genus Actinomyces has been documented as one of the most diverse in the family Actinomycetaceae, order Actinomycetales, class Actinomycetes of phylum Actinomycetota (Yassin 2014;Oren and Garrity 2021). The genus consists of a heterogeneous group of Gram-stain-positive, obligately or facultatively anaerobic, non-spore-forming, non-motile, rodshaped bacteria rich in G + C content (Goodfellow 2012). Despite being reported in 1877 (Harz 1877) and reports of more than 200 child taxa, 33 species have been published validly to date (https:// lpsn. dsmz. de/ genus/ actin omyces). The genus is reported from diverse habitats, including earlier reports on the isolation of pathogenic species from clinical specimens of humans and animals (Funke et al. 1997;Woo et al. 2003;Vielkind et al. 2015). However, several recent The GenBank accession number for the 16S rRNA gene sequence of CtC72 T is KX553980. Additionally, the accession number for the whole genome sequence of the strain CtC72 T is NZ_ MTPX02000000 (assembly accession number: GCA_002154335.2). Contents Category: New Taxa (Actinobacteria).
1 3 9 Page 2 of 11 reports are from rumen or faecal samples of herbivores (An et al. 2006;Palakawong et al. 2016;Zhu et al. 2020;Yang et al. 2021). The gut ecosystem of herbivores is characterised by a diverse population of obligately anaerobic microbes, predominantly bacteria, followed by archaea, protozoa, and fungi (Sirohi et al. 2012;Sengupta et al. 2022). Among bacteria, the cellulolytic and hemicellulolytic ones, including the genus Actinomyces, play a crucial role in making the lignocellulosic feed available to the host as organic acids (An et al. 2006;Palakawong et al. 2016). Such cultures, therefore, can convert abundantly available lignocellulosic biomass into industrially important chemicals or biofuels. Previously, we had also reported the isolation and genomic analysis of cellulose and xylan degrading Actinomyces sp. strain CtC72 T from cattle rumen (Joshi et al. 2018). The present study reports the detailed physiological, biochemical, chemotaxonomic, phylogenetic, and genomic characteristics of strain CtC72 T . Based on the polyphasic taxonomic characterization, we propose that the strain be classified as a new species of the genus Actinomyces and propose the name Actinomyces ruminis sp. nov.

Morphological and physiological characterization
The strain CtC72 T was grown in Hungate roll tubes (Hungate 1969) on PYGR agar (Holdeman et al. 1977) or broth medium for two days at 37 ℃ ± 1 ℃ (pH 7.0 ± 0.1) and evaluated for the colony and cellular morphology (Lanjekar et al. 2015). The colony shape, size and colour were examined using a stereomicroscope (Leica M205 FA, Danaher, Germany). The reaction to Gram-staining was assessed as per the manufacturer's instructions (K001, Hi-Media, India) using a bright-field microscope (Eclipse 80i, Nikon, Japan). The cell motility was examined using phase-contrast microscopy (Eclipse 80i, Nikon, Japan). The cell shape and size were documented using scanning electron microscopy (Zeiss EVO MA15, Carl Zeiss AG, Germany) and expressed as the mean width and length of 10 cells.
The optimum growth conditions of temperature, pH, salinity, and ability to grow in the presence of air were determined in triplicate using PYGR broth as a growth medium and observed for five days, measuring the A 600 of the culture. Growth of the strain CtC72 T at different temperatures of 15-55 ℃ (at intervals of 5 ℃), pH range of 5.0-10 (at intervals of 0.5 pH unit, adjusted with 1 M HCl or NaOH) and NaCl concentrations of 0-3% (w/v, at intervals of 0.5%) was assessed. The ability of strain CtC72 T to grow in the presence or absence of air was examined by incubating the cultures under aerobic or anaerobic conditions (Holdeman et al. 1977). The generation time of the strain was determined by monitoring the A 600 every 1 h up to 72 h under optimised conditions.

Molecular characterization and phylogenetic analysis
The DNA isolation and generation of the 16S rRNA gene sequence of the strain CtC72 T has been documented in our earlier study (Joshi et al. 2018), submitted to GenBank under the accession number KX553980. The 16S rRNA gene sequence-based similarity searches were performed using NCBI GenBank BLASTn function. The G + C content of the DNA was determined by monitoring fluorescence intensity during DNA denaturation in the capillary tube (Xu et al. 2000) at the identification and characterization service of the National Centre for Microbial Resource (Pune, India).
For phylogenetic analysis, sequences of closely related species of the genus Actinomyces were downloaded from the NCBI GenBank database and aligned using MAFFT (Katoh et al. 2019). The aligned sequences were used to construct phylogenetic trees using the Maximum-Likelihood (ML) method in W-IQ-TREE (Trifinopoulos et al. 2016), the Neighbour-Joining (NJ) method using NGPhylogeny. fr server (Lemoine et al. 2019) and the Maximum-Parsimony (MP) method in the MEGA11 (Tamura et al. 2021). Bootstrap resampling analysis (Felsenstein 1985) of 1000 replicates was performed to estimate the confidence of tree topologies. The genus Bifidobacterium bifidum KCTC 3202 T (GenBank accession number: NR_044771) was used as an outgroup, and all the trees were visualized using iTOL v5 (Letunic and Bork 2021).

Ecological distribution
Being a single species strain, the occurrence of the strain CtC72 T was determined in other environmental habitats. For this, the 16S rRNA gene sequence of strain CtC72 T was queried with the GenBank dataset. All the closely related cultured and uncultured sequences were downloaded, and their phylogenetic positions were inferred using a maximum-likelihood-based phylogenetic tree, as described earlier. Next, we documented the ecological distribution of the nearest clades from the GenBank dataset.
Additionally, the 16S rRNA gene sequence and the genome of strain CtC72 T were uploaded to the Protologger server (Hitch et al. 2021) for ecological analysis. In Protologger, each 16S rRNA gene sequence is compared to 19,000 amplicon samples representing a total of 38,163,501 OTUs (operational taxonomic units) obtained from 19 environments within the IMNGS database (Lagkouvardos et al. 2016). Sequence comparison is conducted using blastN with > 80% OTU coverage, > 97.0% identity, and e-value < 1e-25.

Genome sequences analysis for taxonomic and functional features
The whole genome of the strain CtC72 T was previously sequenced and submitted to GenBank under the accession number NZ_ MTPX02000000 (Joshi et al. 2018).
The taxonomically important genomic features of the strain CtC72 T with the nearest phylogenetic affiliates were compared to ascertain its novelty. The phylogenomic analysis of strain CtC72 T and other reported strains of Actinomyces was performed using the Codon Tree method within PATRIC (Davis et al. 2020), which uses cross-genus families (PGFams) as homology groups. A total of 269 PGFams were found among the selected genomes using Codon Tree analysis, and the aligned proteins and coding DNA from singlecopy genes were used for RAxML analysis. The genus Bifidobacterium bifidum ATCC 29521 T was used as an outgroup (PATRIC accession number: 500634.4).

Biochemical and chemotaxonomic characterization
All the biochemical tests like substrate utilization, glucose fermentation, metabolic end-product analysis, growth in 20% (w/v) bile (using Oxgall), esculin hydrolysis, presence of oxidase, catalase, urease, reduction of nitrate were carried out in triplicates by the method described by Holdeman et al. 1977. Additional biochemical characteristics were determined using Biolog AN MicroPlate™ as per the manufacturer's instructions (Biolog Catalog 1007). Metabolic products of glucose fermentation were analysed using a gas chromatograph (GC) and high-performance liquid chromatograph (HPLC) systems. The concentration of volatile fatty acids (VFAs) like acetic acid, propionic acid, butyric acid, valeric acid, caproic acid, etc., was determined using GC (Chemito 8610, India) equipped with Flame Ionization Detector and IRIS 32 software. The oven temperature was kept at 150 °C, the injector at 170 °C, the detector at 190 °C, column used was Chromosorb W (HP) (1.83 m × 3.2 mm SS) packed with 10% FFAP and 2% H 3 PO 4 . The carrier gas used was N 2 at the flow rate of 30 ml min −1 . The fermentation gases like hydrogen and carbon dioxide were analysed on GC (PerkinElmer, Autosystem XL) equipped with a thermal conductivity detector and TotalChrom Navigator software. The oven temperature was kept at 40 °C, the injector at 70 °C, the detector at 100 °C, the column used was Porapak Q (1.83 m × 3.2 mm), and the carrier gas was Argon at 40 ml min −1 . The presence of non-VFAs like formic acid, lactic acid, succinic acid, malic acid, etc., was tested on HPLC-LC20A (Shimadzu, Japan) equipped with a Refractive Index detector, SIL-20AC HT autosampler, and Lab solutions software. The oven temperature was kept at 40 °C, the column used was Aminex HPX-87H (300 × 7.8 mm) (Biorad, USA), and the mobile phase was 5 mM H 2 SO 4 at a flow rate of 0.7 ml min −1 .
All the chemotaxonomic analyses were outsourced to the identification & characterization service of the National Centre for Microbial Resource (Pune, India). The strain CtC72 T was grown in PYGR broth under optimum growth 9 Page 4 of 11 conditions for 48 h and processed for the cellular fatty acid profile using Sherlock Microbial Identification System (MIDI, 2001) (Sherlock 1999) and MALDI-TOF-MS based protein profile using MALDI Biotyper (Pandey et al. 2019).

Morphological and physiological characteristics
The comparison of various characteristics of strain CtC72 T and its nearest relatives are detailed in Table 1. Colonies of strain CtC72 T were 2.0-3.0 mm in diameter, circular in shape, and white in colour. The cells of strain CtC72 T were non-spore forming, Gram-stain positive, non-motile rods having a size ranging from 0.6-1.0 × 2.6-4.0 µm (Fig. S1). The strain CtC72 T grew between temperatures of 30-50 °C; pH range between 6.0 and 9.0, and NaCl concentrations between 0 and 1.5%, but the optimum growth was achieved at 40 °C and pH of 7.5-8.0 in the absence of NaCl. The strain CtC72 T grew only under anaerobic conditions, and no growth was observed under aerobic conditions, indicating its obligate anaerobic nature. The mean generation time for strain CtC72 T was 2.7 h when grown under optimum conditions.

Molecular and phylogenetic characteristics
The 16S rRNA gene sequence similarity results (Table 1) showed that the strain CtC72 T shared the highest sequence similarity with A. succiniciruminis TISTR 2317 (98.49%), A. glycerinitolerans TISTR 2318 (98.41%), A. qiguomingii DSM 106201 T (98.30%), A. ruminicola DSM 27982 T (97.49%) and with A. procaprae JCM 33484 T (97.18%). The 16S rRNA gene sequence similarity values of less than 98.65% (Kim et al. 2014) to all other validly recognised Actinomyces species, highlights the novel nature of strain CtC72 T . The wet lab (T m ) based DNA G + C content (in mol%) of strain CtC72 T was 68.4, within the same range as reported for other related Actinomyces species (Table 1).
The different phylogenetic trees also indicated the identity of strain CtC72 T as a member of the genus Actinomyces. Further, a separate clade supported by a high bootstrap value showed its novelty and distinct lineage from its closest phylogenetic neighbour A. qiguomingii DSM 106201 T . The ML method confirmed the statistical consistency and accuracy of the evolutionary relationship of our strain (Fig. 1). These findings were also true for evolutionary distance-based NJ (Fig. S2) and character-based MP (Fig. S3) trees (Taboada-Echalar et al. 2013). Therefore, these phylogenetic analyses confirmed that the strain CtC72 T could be classified as a new species within the genus Actinomyces.

Ecological distribution
The phylogenetic affiliates of strain CtC72 T were also obtained from the rumen of cattle like A. succiniciruminis TISTR 2317 (Netherlands), and A. ruminicola DSM 27982 T (China) or rumen of sheep (Netherlands) like A. glycerinitolerans TISTR 2318. The other affiliates, viz. A. qiguomingii DSM 106201 T and A. procaprae JCM 33484 T were also of herbivorous gut origin but cultured from faeces of Tibetan antelope and Tibetan gazelle (China), respectively.
The subsequent ecological distribution study also revealed ≤ 99% sequence similarities of strain CtC72 T in the GenBank dataset with several cultured (both type and non-type strains) and non-cultured representatives of genus Actinomyces, but none showed 100% similarity (Fig. S4). This may be due to the recent shift of the scientific community towards targeted metagenomics bypassing cloning methods. Nevertheless, the nearest matches of the strain CtC72 T were with non-type strains from the rumen or faeces of herbivores, mainly buffalo, cattle, red deer, sheep, Tibetan gazelle, and Tibetan antelope (96%-99%), indicating the strain's possible presence in gut environments.
Surprisingly, no MAGs matching our strain's genome could be identified from 49,094 MAGs obtained from 10 environments, including permafrost, primates, groundwater, soil, ocean, hydrothermal sediment, human, pig, mouse and rumen. Our results, therefore, suggest the presence and distribution of cultures similar to strain CtC72 T in gut environments, the absence of similar MAGs probably indicates its limited abundance.

Genomic characteristics
The genome of strain CtC72 T has been reported in our previous study to contain genes related to the production of carbohydrate-active enzymes, ethanol, stress tolerance, chitin degradation, pectin utilization, sugar utilization, etc. (Joshi et al. 2018). The detailed analysis and comparison of various taxonomically important genomic features too revealed the distinct nature of strain CtC72 T . The phylogenomic tree also showed the strain CtC72 T as Actinomyces affiliate nearest but distinct to the five closest members, further affirming its novelty (Fig. 2). The digital G + C content (in %) of strain CtC72 T was 68.05, equivalent to its wet lab (T m ) value (Table 1). The genome size and digital G + C content of strain CtC72 T were also similar to its nearest phylogenetic  (30) pH range (optimum) 6.0-9.0 (7.5-8.0) 6.0-9.0 (7) 6.5-9.0 (8.0-8.5) 6.0-9.0 (7) 5.5-8.5 (7.5) 5.5-8.5 (7.5) affiliates ( Table 1). The dDDH value of strain CtC72 T was between 37 and 62% with all of its compared neighbours (Table 1), which is substantially below the threshold value of 70% proposed for designating a new species. Similarly, the ANI and AAI values between the strain CtC72 T and related members of genus Actinomyces specified shared values of < 90% (Table 1), much less than the recommended cut-off values of 95% for delineating new bacterial species (Chun et al. 2018). The detailed genomic comparison between different taxonomically important indexes strongly advocated that strain CtC72 T be classified as a novel Actinomyces species.

Biochemical and chemotaxonomic characteristics
Strain CtC72 T fermented glucose majorly to acetic acid (25.93 ± 0.18 mM) and formic acid (26.52 ± 0.34 mM), whereas propionic acid (2.60 ± 0.03 mM), lactic acid (9.63 ± 0.18 mM), ethanol (6.69 ± 0.79 mM), and CO 2 (5.68 ± 0.25 mM) were the minor metabolic end products. The strain CtC72 T did not grow in the presence of 20% (w/v) bile. The strain reduced nitrate but was negative for esculin hydrolysis and catalase, oxidase, and urease production. The carbon source utilization profile generated using Biolog AN plate revealed that the strain CtC72 T could oxidise 36 substrates as the sole carbon source (Table S4), including several dimeric and monomeric substrates. The utilization of several disaccharides indicates the ability of strain CtC72 T to produce hydrolytic enzymes necessary for their breakdown. The presence of genes encoding diverse Carbohydrate-Active enZYmes (CAZymes) in the genome of strain CtC72 T and its ability to degrade polymeric lignocellulose constituents avicel, CMC and xylan has been reported earlier (Joshi et al. 2018). Overall, the strain CtC72 T differed from its closest phylogenetic neighbours in its inability to utilise D-melibiose and hydrolyse esculin. Due to the varying optimal growth conditions (Table 1), cellular fatty acid profiles of all strains were not performed in parallel under same cultural conditions. Barring the obligately anaerobic strain CtC72 T (40 °C, 48 h) and A. ruminicola DSM 27982 T (An et al. 2006), which were grown in PYGR medium, A. qiguomingii DSM 106201 T (37 °C) and A. procaprae JCM 33484 T (35 °C, late exponential phase) were grown in brain heart infusion-sheep blood medium aerobically in presence of 5% CO 2 (Zhu et al. 2020;Yang et al. 2021), whereas oxygen tolerant anaerobic strains A. succiniciruminis TISTR 2317 and A. glycerinitolerans TISTR 2318 were cultured (37 °C, 48 h) in modified bicarbonatebuffered anaerobic medium (Palakawong et al. 2016). The major (> 10%) cellular fatty acids of strain CtC72 T were identified as C 16:0 (24.9%), C 18:1 ω9c (19.6%) and C 18:1 ω9c DMA (17.1%), as detailed in Table S5. The comparison of cellular fatty acids based on published data revealed differences in proportions of C 16:1 ω7c, C 18:1 ω7c, C 18:1 ω9c DMA, and C 19:0 iso (Table S5), further highlighting separate lineage for strain CtC72 T .
The MALDI Biotyper profile of strain CtC72 T showed a complex pattern of a well-defined spectrum with over 15 peaks in m/z ratio from 2616 to 9615 (Fig. S5). The comparison of MALDI Biotyper profiles of strain CtC72 T with closely related type strains showed several unique peaks (4267, 4807, 6344, 6742 and 7739), differentiating the newly isolated strain (Table S6). Only a single peak around the m/z ratio of ~ 5880 in strain CtC72 T was similar to Actinomyces ruminicola DSM 27982 T . However, these exclusivities cannot be fully established due to the lack of MALDI Biotyper profile data for Actinomyces qiguomingii DSM 106201 T and Actinomyces procaprae JCM 33484 T .

Taxonomic conclusion
The physiological, phylogenetic, genomic, biochemical and chemotaxonomic characteristics reveal that the strain CtC72 T belongs to the genus Actinomyces but is distinct from the currently known species. Also, this strain adds to the list of obligately anaerobic members of the genus Actinomyces. Therefore, the strain CtC72 T should be classified as a novel species for which the name Actinomyces ruminis sp. nov. is proposed.